Structure of the N-linked oligosaccharide of the main diagnostic antigen of the pathogenic fungus Paracoccidioides brasiliensis

The major diagnostic antigen of Paracoccinioides brasiliensis is the exocellularly secreted 43,000 Da glycoprotein (gp43) which contains a single N-linked oligosaccharide chain, This oligosaccharide, although poorly immunogenic in man, is responsible for the cross-reactivity of the gp43 with sera from patients with histoplasmosis, and may have a role in fungal virulence, It contains a neutral high-mannose core (Man(7)GlcNAc(2)) to which a (1-->6)-linked alpha-D-Manp chain of variable length, substituted at the 2-O positions by single alpha-D-Manp residues, is attached, A terminal unit of beta-D-galactofuranose is (1-->6)-linked to one of the (1-->2)-linked mannosyl residues, either in the C or in the A arm of the oligosaccharide, The heterogeneity of the oligosaccharide is determined by the different sizes of the A arm and the sites of insertion of the beta-galactofuranosyl unit. The complete structure was determined hy methylation analysis, H-1-NMR, mass spectrometry, acetolysis and mannosidase degradation, Electrospray mass spectrometry showed that the oligosaccharide comprises several subtypes ranging from Hex(18)GlcNAc(2) to Hex(10)GlcNAc(2) which accounts for the diffuse migration of the gp43 in polyacrylamide gels, The average size of the most frequent subtype is Hex(13.6)GlcNAc(2). Dilute acid treatment to remove beta-D-Galf reduced the molecular masses of the majority of the subtypes by a single sugar unit.